Article Figures & Data
Figures
- Figure 1.
Differentiating and characterizing iPSC-derived neurons. A, Timeline of iPSC culture and differentiation from day 0 (D0) to NPCs by D20, and subsequent differentiation to forebrain NPCs (D25) and forebrain neurons (D55). B, iPSCs express the pluripotency marker OCT4 (green). C, D, NPCs express the neural lineage markers NESTIN (green), PAX6 (red), and SOX2 (red). E, Differentiated neuron cultures stained for tubulin (green). F, Terminally differentiated neurons express the presynaptic protein synapsin (green) and dendritic protein MAP2 (red). G, Terminally differentiated neurons express the axonal protein Tau (blue) and the presynaptic protein SV2 (green).
- Figure 2.
Validation of GCase inhibition with CBE. A, B, Relative to controls, 10-d CBE-treated neurons incubated with the GCase substrate GBA1-FQ6 show negligible fluorescence activity. C, Relative fluorescence units (RFU) per cell. N = 3 independent experiments run in duplicate; Control, n = 19 fields of view; CBE, n = 21 fields of view. CBE treatment (100 μm) significantly reduces GCase activity relative to controls (*p < 0.0001; Extended Data Table 1-1). D, Ten days of 100 μm CBE treatment significantly upregulates the staining intensity of astrocytic marker GFAP compared with controls. E, Average GFAP staining intensity per field of view. N = 3 independent experiments run in duplicate; Control, n = 112 fields of view; CBE, n = 120 fields of view. Average GFAP staining intensity per field of view is significantly increased in CBE-treated cultures (*p < 0.0001; Extended Data Table 1-1).
- Figure 3.
CBE does not affect axonal transport of LAMP1-GFP vesicles. 54 DIV neurons were transfected with the lysosome marker LAMP1-GFP and imaged 24 h later. A, Still frames of a representative axon from a control video sampled at various time points (Movie 1) and (B, C) representative kymographs from one control and one CBE-treated LAMP1-GFP video. Red arrows denote retrograde; blue arrows denote anterograde. Videos were acquired at 1.5 frames/s for 120 s. D, E, Analysis of kymographs revealed no significant effect of CBE treatment on transport parameters (N = 4 independent experiments run in duplicate; Control, n = 116 cells, 2080 transport events; CBE, n = 109 cells, 2179 transport events, n.s. = nonsignificant, p > 0.05). F, Table of all measured transport parameters (±SD) and Extended Data Table 1-1.
- Figure 4.
CBE does not affect axonal transport of acidic vesicles. 55 DIV neurons were stained with LysoBrite and imaged live. A, Still frames of one representative axon from a control condition transport video sample at various time points and (B, C) representative kymographs from one control and one CBE-treated LysoBrite transport video. Red arrows denote retrograde; blue arrows denote anterograde. Videos were acquired at one frame per second for 90 s. D, E, Analysis of kymographs revealed no significant effect of CBE treatment on any transport parameter (N = 3 independent experiments run in triplicate; Control, n = 116 cells, 752 transport events; CBE, n = 140 cells, 1180 transport events, n.s. = nonsignificant, p > 0.05). F, Table of all measured transport parameters (±SD) and Extended Data Table 1-1.
- Figure 5.
CBE does not affect axonal transport of mitochondria. 54 DIV neurons were transfected with mitochondrial marker MTS-dsRed2 and imaged 24 h later. A and Movie 2, Still frames of a representative axon from a control condition video at various time points and (B, C) representative kymographs from one control and one CBE-treated MTS-dsRed2 video. Red arrows denote retrograde; blue arrows denote anterograde. Videos were acquired at 0.5 frames/s for 180 s. D, E, Analysis of kymographs revealed no significant effect of CBE treatment on any transport parameter (N = 3 independent experiments run in triplicate; Control, n = 40, 437 transport events; CBE, n = 35, 470 transport events, n.s. = nonsignificant, p > 0.05). F, Table of all measured transport parameters (±SD) and Extended Data Table 1-1.
- Figure 6.
CBE does not influence lysosomal rupturing following LLoME treatment. CBE-treated NPCs and controls assessed for lysosomal rupture events by staining for LAMP1 (top, red) and p62 (middle, green), and colocalized (bottom). A, B, In the absence of LLoME, neither CBE nor control NPCs develop p62-positive lysosomes. C, D, Following treatment of 600 μm LLoME for 1 h, p62 puncta form in both CBE and control conditions. E, p62-positive puncta colocalizing with LAMP1 puncta were analyzed using the ImageJ plugin ComDet, revealing no significant difference in puncta relative fluorescence units (RFU) per cell. F, The percent of LAMP1 positive puncta that colocalized with p62 puncta, indicating lysosomal rupture, per cell were analyzed using ComDet. There was no significant difference in the percent ruptured lysosomes per cell between controls and CBE-treated NPCs (N = 3 independent experiments run in duplicate; Control +LLoME, n = 257 cells; Control −LLoME, n = 91 cells; CBE +LLoME, n = 201 cells; CBE −LLoME, n = 91 cells; Extended Data Table 1-1).
Movies
- Movie 1.
Live imaging of axons showing LAMP1-eGFP transport in control versus CBE-treated cells. 1.25 frames/s, 20-s recording, playback: 1× real time.
- Movie 2.
Live imaging of axons showing MTS-dsRed2 transport in control versus CBE-treated cells. 0.5 frames/s, 90-s recording; playback: 4× real time.
Extended Data Figure 1-1
CBE does not influence LC3 staining following LLoME treatment. CBE-treated NPCs and controls assessed for lysosomal rupture events by staining for LAMP1 (top, red) and LC3 (middle, green), and colocalized (bottom). A, B, In the absence of LLoME, neither CBE nor control NPCs develop LC3-positive lysosomes. C, D, Following treatment of 600 μm LLoME for 1 h, LC3 puncta form in both CBE and control conditions. E, LC3-positive puncta colocalizing with LAMP1 puncta were analyzed using the ImageJ plugin ComDet, revealing no significant difference in puncta relative fluorescence units (RFU) per cell. F, The percent of LAMP1 positive puncta that colocalized with LC3 puncta, indicating lysosomal rupture, per cell were analyzed using ComDet. There was no significant difference in the percent ruptured lysosomes per cell between controls and CBE-treated NPCs (N = 3 independent experiments run in duplicate; Control +LLoME, n = 90 cells; Control −LLoME, n = 32 cells; CBE +LLoME, n = 68 cells; CBE −LLoME, n = 36 cells; Extended Data Table 1-1). Download Figure 1-1, TIF file.
Extended Data Table 1-1
Student t test p values for all data. Download Table 1-1, DOC file.
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